Streptomyces sp. RI24 was isolated from a soil sample collected at Shuri, Okinawa Prefecture, Japan by the sodium dodecyl sulfate-yeast extract method.7 The strain was cultivated in 50-ml test tubes, each of which contained 15 ml of a seed medium consisting of starch (Kosokagaku, Tokyo, Japan) 1.0%, polypeptone (Nihon Pharmaceutical, Tokyo, Japan) 1.0%, molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan) 1.0% and meat extract (Extract Ehlrich; Wako Pure Chemical Industry, Osaka, Japan) 1.0%, pH 7.2 (before sterilization). The test tubes were shaken in a reciprocal shaker (355 r.p.m.) at 27 °C for 2 days. Aliquots (2.5 ml) of the seeding culture were transferred to 500-ml baffled Erlenmeyer flasks containing 100 ml of a production medium that contained starch (Kosokagaku) 2.5%, soybean meal (Nisshin Oillio, Tokyo, Japan) 1.5%, dry yeast (Mitsubishi Tanabe Pharma, Osaka, Japan) 0.2%, CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan) 0.4% and Diaion HP-20 resin (Mitsubishi Chemical, Tokyo, Japan) 1.0%, pH 7.4 (before sterilization), and then cultured in a rotary shaker (180 r.p.m.) at 27 °C for 5 days.

The mycelial cake was separated from the fermentation broth (2 liters) by centrifugation, followed by extraction with acetone (400 ml). After being concentrated in vacuo, the residual aqueous concentrate was extracted with EtOAc (100 ml, × 3). The organic layer was dried over Na2SO4 and then allowed to evaporate under reduced pressure until thoroughly dry. The dried residue (770 mg) was reconstituted in CHCl3 (1 ml) subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60 column packing with 30-g silica gel; Shoko Scientific, Tokyo, Japan) using a CHCl3–MeOH stepwise solvent system (0, 2 and 5% MeOH). The 2% MeOH eluate (38 mg) was further purified by preparative reversed-phase HPLC using an L-column 2 ODS column (20 i.d. × 150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan) with a 2996 photodiode array detector (Waters, Milford, MA, USA) and a 3100 mass detector (Waters), using 70% aqueous MeOH containing 0.1% formic acid (flow rate, 10 ml min−1) as a developing solvent to yield 1 (11.6 mg, retention time (Rt) 27.3 min). Likewise, CH3Cl eluate (17 mg) from silica gel chromatography was purified by preparative reversed-phase HPLC that was developed using 75% aqueous MeOH containing 0.1% formic acid to yield 2 (1.1 mg, Rt 33.6 min).

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